Our goal during the past thirty years of NIH funding has been to understand the mechanism of chemotaxis in the Gram-positive bacterium Bacillus subtilis. During the past decade, the realization has emerged that the B. subtilis paradigm might be the ideal one for understanding these diverse mechanisms in the broad sweep of bacteria and archaea because of its inclusion of most known chemotaxis proteins and its similarity to the mechanism used in many archaea and the primitive thermophile Thermotoga maritima. This similarity suggests that the B. subtilis mechanism is an ancient mechanism and close to the progenitor mechanism that existed just before the divergence of bacteria and archaea. Thus, in studying this mechanism, we can understand how chemotaxis in bacteria and archaea might have evolved and gain insights into how these diverse pathways may function. During the last funding period, we have made tremendous progress in elucidating the biochemical function of the three proteins not found in Escherichia coli chemotaxis: CheC, CheD, and CheV. Collectively, this work has enabled us to formulate a working hypothesis for how the integrated pathway functions. The key element of this hypothesis is that there are three adaptation systems in B. subtilis (unlike just one in E. coli). In addition to the previously identified methylation and CheV systems, we have discovered a third adaptation system involving CheY, CheC, and CheD. The available evidence indicates that CheD is an allosteric activator of the receptor complex. Furthermore, we now have a molecular model for how the selective methylation of specific residues on the receptors differentially affects CheA kinase activity, a key element of which is a newly discovered zinc ion in the methylation region. These breakthroughs have been greatly facilitated by the recent structures for T. maritima CheC, CheD, and the cytoplasmic domain of TM1143 (a chemoreceptor). As a result, we are now in a position to propose detailed experiments to elucidate key mechanisms in B. subtilis chemotaxis. Specifically, we wish to develop a computational model for the integrated pathway and test various aspects experimentally. We will primarily focus on selective methylation and CheD binding, determining how specific methylation sites affect CheA kinase activation and CheD binding and how CheD binding affects CheA kinase activation and methylation. We will also focus on the relation between the CheC/D/Yp and CheV adaptation systems and the very slow receptor remethylation process. The proposed experiments include (among others): (i) measuring timing of methylation at each site in response to the addition and removal of attractants in McpB (weak CheD requirement) and McpC (strong CheD requirement) in strains having the CheV and CheC/D/Yp adaptation systems and lacking one or both;(ii) determining the role of each methylated site on CheD binding, CheA activation, and on receptor structure as reflected by disulfide crosslinking;and (iii) discovering where CheD binds and how CheD binding affects receptor methylation and CheA kinase activity. PUBLIC HEALTH RELEVANCE: Chemotaxis is a virulence factor in many pathogens, so we can safely assume that the design of antibiotics that target chemotaxis and motility will be facilitated by understanding the B. subtilis chemotaxis mechanism. Furthermore, many Gram-negative pathogens including Burkholderia sp., Campylobacter jejuni, Helicobacter sp., and Treponema pallidum have homologues to CheC, CheD, and CheV, genes first identified and then characterized in B. subtilis.